Project Summary/Abstract Vitamin A is an essential nutrient that mediates a variety of biological processes including immunity, embryogenesis and vision. Alterations of vitamin A metabolism have been implicated in numerous developmental and genetic defects, as well as in various pathological states in adults, including blindness, skin disease and cancer. Retinoids (vitamin A metabolites) circulate in the bloodstream in the form of retinol (ROH) bound to retinol-binding protein (RBP). The transmembrane protein, STRA6 (Stimulated by Retinoic Acid 6) is expressed in blood-tissue barriers, especially the retinal pigment epithelium (RPE) and mediates cellular uptake from ROH-bound RBP and efflux of ROH to apo-RBP from the cell. Mutations in STRA6 have been shown to cause Matthew Wood Syndrome, which is characterized by severe congenital malformations such as microphthalmia and anophthalmia in combination with pulmonary hypoplasia. The Mancia lab has recently solved the structure of STRA6 by single particle cryo-electron microscopy (cryo-EM). The protein is assembled as an obligate dimer with novel topology; unexpectedly, each protomer is tightly associated with one molecule of calmodulin (CaM) on the cytoplasmic side. This interaction is physiological and suggests that cellular Ca2+ levels and STRA6 function might be correlated. Given the tight control of vitamin A and [Ca2+] required for phototransduction, we hypothesize that ROH uptake into the RPE is modulated by intracellular Ca2+ levels. Illumination of the retina catalyzes both turnover of 11-cis-retinal and a net efflux of Ca2+ from the photoreceptor cells towards the RPE. Excessive uptake of ROH in response to 11-cis-retinal turnover, which can be toxic, could be prevented by a signal from the released Ca2+. A conformational change in STRA6 when in complex with Ca2+-CaM vs. apo-CaM may translate to changes in affinity of STRA6 for apo- versus holo- RBP, thus promoting one direction of ROH flux over the other. To address these questions, proteoliposome and cell-based assays of ROH uptake and RBP-STRA6 binding in addition to CaM/STRA6 complex structural analysis will be utilized. Two aims are proposed: (1a) to investigate the influence of intracellular Ca2+ on STRA6-mediated retinol uptake and efflux, (1b) to study the importance of Ca2+ in RBP-STRA6 interaction and (2) to compare STRA6 conformation in the presence of Ca2+-bound CaM and apo-CaM. Understanding the relationship between Ca2+ and vitamin A transport by STRA6 at the molecular level is imperative, as it may provide new insight into phototransduction and potential treatments of ocular disease.